Nanoscale Zeolitic Imidazolate Framework-8 for Ratiometric

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Nanoscale Zeolitic Imidazolate Framework-8 for Ratiometric Fluorescence Imaging of MicroRNA in Living Cells Jin-Tao Yi, Ting-Ting Chen, Jia Huo, and Xia Chu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03369 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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Analytical Chemistry

Nanoscale Zeolitic Imidazolate Framework-8 for Ratiometric Fluorescence Imaging of MicroRNA in Living Cells

Jin-Tao Yi, Ting-Ting Chen, Jia Huo* and Xia Chu*

State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China.

E-mail: [email protected]; [email protected]

*

Corresponding

author.

Tel.:

86-731-88821916;

Fax:

[email protected]; [email protected].

1 Environment ACS Paragon Plus

86-731-88821916;

E-mail:


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ABSTRACT: MicroRNAs (miRNAs) play important roles in cell differentiation, proliferation and apoptosis and have been recognized as valuable biomarkers for clinical disease diagnosis. Here, we adopt for the first time zeolitic imidazolate framework-8 (ZIF-8) as a nanocarrier to efficiently deliver nucleic acid probe to living cells and develop a novel ratiometric fluorescence strategy based on DNAzyme for miRNA-21 imaging. A Cy5-labeled 8-17 DNAzyme strand and a Cy3-labeled substrate strand containing a complementary segment to the target miRNA-21 first form duplex probe and fluorescence resonance energy transfer (FRET) takes place. After adsorption on the ZIF-8 surface and cellular uptake, the probe/ZIF-8 nanocomplex degrade in acidic endosome and release duplex probes and Zn2+, and the latter can act as an effective cofactor for 8-17 DNAzyme. The intracellular miRNA-21 hybridizes with the complementary segment of the substrate strand and results in dissociation from DNAzyme-substrate duplex probe after DNAzyme cleaves the substrate to two fragments, accompanied with the change in the FRET signal. The proposed method has been applied to image miRNA-21 expression levels in MCF-7, HeLa and L02 cells with high contrast and reliability. The fluctuation of miRNA-21 expression level induced by miRNA-21 mimic or inhibitor can also be monitored through the obvious imaging color change. Taken together, the proposed method provides a powerful tool for cancer diagnosis and miRNA-associated biological study.


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INTRODUCTION MicroRNAs (miRNAs) are highly conserved noncoding, small single-stranded endogenous RNAs with the length of 19-23nt and play important roles in several biological processes such as cell differentiation,1 proliferation2 and apoptosis.3 More and more researches have demonstrated that disordered expression of miRNAs is closely related to many diseases, including diabetes, neurological diseases, viral infections, stroke induced tissue injury and human cancers.4-6 Therefore, miRNAs can be used as valuable biomarkers for clinical disease diagnosis and cellular level research. However, the detection of miRNAs remains challenging because of their short size, vulnerable degradability, low expression levels in cells, and high sequence homology among miRNA family members. Traditional detection methods include northern blotting,7-9 quantitative fluorescence reverse transcription PCR (RT-PCR)10-12 and microarrays.13 Northern blotting is a semiquantitative detection method that frequently suffers from time and labor consumption combined with low sensitivity. RT-PCR has merits of high detection sensitivity and good specificity, but this method needs reverse transcription to cDNA prior to the amplification step, causing problems in performing highly parallel qRT-PCR from sequence-specific differences during primer annealing. Microarray technology can detect multiple miRNA samples in a short time, but it has poor reproducibility and low sensitivity. Recently, many other methods have been emerged to detect miRNAs, such as mass sepectrometry,14 Raman spectroscopy,15 electrochemiluminescence,16 and nuclease-assisted target recycling.17 Although these methods have good sensitivity, most of them not only need complicated and advanced instrumentation, but also can not satisfy the requirement for the detection of miRNAs in living cells.



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To realize the intracellular detection of miRNAs, approaches that can efficiently deliver the detection probe to cytoplasm of the living cells are required. Because the commonly used nucleic acid-based probe can’t penetrate the cell membrane, various nanomaterials are generally utilized to deliver DNA probe through endocytosis. Recently, many delivery strategies based on nanomaterials have been successfully applied to monitoring intracellular miRNA levels in situ. Gold-upconversion nanoparticle pyramids were fabricated to quantify miRNA level in living cells using plasmonic circular dichroism and luminescence dual-mode.18 Gold-based nanomaterials (nanoparticles or nanorods)19-23 and silica nanoparticles24,25 have been employed to deliver nucleic acid probe through Au-SH assembly or NHS-mediated covalent crosslink. In addition, graphene-based nanosheets26 or quantum dots27,28 and MnO2 nanosheets29 were also popular because of their strong adsorption toward single-stranded nucleic acid. Despite these successes, the probes immobilized on the surface of nanomaterials through Au-SH assembly or covalent crosslink can’t dissociate from nanomaterials after internalizing into cells. Graphene or MnO2 nanosheets are only suited to deliver single-stranded nucleic acid probe. In addition, most of nanomaterials can’t be biodegraded in the cells. These drawbacks limit their further applications in the nucleic acid-based assay in living cells. Metal-organic frameworks (MOFs) are an emerging class of functional materials with intriguing features such as high surface area, tunable shapes and pore sizes, and controllable surface functionalities, and have been exploited in a wide range of applications including gas storage and separation, selective catalysis, drug delivery and sensing.30-35 Different from most MOFs, zeolitic imidazolate framework-8 (ZIF-8), a MOF built from zinc ions and 2methylimidazole, possesses unique pH-sensitive degradation property due to the effects of


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protonation, and a number of pH-responsive drug delivery systems have been developed by taking advantage of this attractive property.36-40 Because of the positively charged Zn2+ on the surface of ZIF-8, we surmise that ZIF-8 can bind nucleic acid probes through electrostatic interaction and coordination between phosphate residues on the nucleic acid backbone and vacant Zn sites on the ZIF-8 surface. In addition, the positively charged Zn2+ can also make nanoparticles easily adsorb at the negatively charged cell membrane, facilitating efficient cellular uptake of the ZIF-8. As a result, ZIF-8 represents a unique nanocarrier platform for efficient delivery of nucleic acid probes. After internalizing into cells, the dissociation of ZIF-8 in acidic endosome releases the nucleic acid probes and Zn2+ ions, and the latter can also serve as an effective cofactor for DNAzyme, enabling the development of DNAzyme-based nucleic acid assay in living cells. DNAzymes are synthetic DNA strands selected by using the SELEX technology, which can catalyze the cleavage of RNA or DNA molecules in a divalent cations-dependent manner. Currently, RNA-cleaving DNAzymes have attracted extensive attentions in metal sensing,41 diagnostic and therapeutic applications.42 In addition, a few attempts have also been made to imaging the metal ions in living cells based on DNAzymes.43,44 Herein, we adopt for the first time ZIF-8 as a nanocarrier to efficiently deliver nucleic acid probe to living cells and develop a novel ratiometric fluorescence imaging strategy based on DNAzyme. ZIF-8 protects nucleic acid probes from nuclease degradation, enhances probe cellular uptake, and promotes probe escape from endosomes. The ratiometric fluorescence imaging strategy based on DNAzyme eliminates the interference from determination conditions and affords a high imaging contrast. This unique



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nanocarrier platform based on ZIF-8 has been utilized successfully to image miRNA-21 expression levels in living cells.

EXPERIMENTAL SECTION Materials

and

Reagents.

Zinc

nitrate

hexahydrate

(Zn(NO3)2•6H2O,

99%),

2-

methylimidazole (C4H6N2, 99%) and bisBenzimide H 33342 trihydro chloride (Hoechast 33342) were purchased from Sigma-Aldrich. Methanol was obtained from Sinopharm Chemical Reagent Company Ltd (Shanghai, China). LysoTracker Green DND-26 was purchased from Invitrogen (Carlsbad, CA). MTT Cell Proliferation and Cytotoxicity Assay Kit were purchased from Beyotime Biotechnology. MicrONTMhsa-miR-21-5p mimic and micrOFFTMhsa-miR-21-5p inhibitors were purchased from RiboBio Company (Guangzhou, China). All other reagents were of analytical grade. Ultrapure water was obtained through a Millipore Milli-Q water purification system (Billerica, USA), which had an electric resistance >18.25 MΩ. The DNA oligonucleotides were synthesized and purified by Sangon Biological Engineering Technology & Company Ltd. (Shanghai, China), whose sequences were shown in Table S1. Apparatus.

The

fluorescence

spectra

were

recorded

using

a

FluoroMax-4

Spectrofluorometer (HORIBA Jobin Yvon, Inc., NJ, USA). Both excitation and emission slit were set at 5.0 nm with a 900 V PMT voltage. Transmission electron microscope (TEM) was performed on a field emission high resolution 2100F transmission electron microscope (JEoL, Japan) operating at an acceleration voltage of

200KV. Zeta potential and dynamic light

scattering (DLS) were measured on the Malvern Zetasizer Nano ZS90 (USA) at room temperature. The crystal phase of ZIF-8 was identified by D8-advance X ray diffraction (Bruker,


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Germany). The concentration of Zn2+ was measured by inductively coupled plasma mass spectrometry (ICP-MS NexION300×, USA). The cell viability was evaluated by a microplate reader (ELx800, BioTek, USA). All fluorescence images were measured on a confocal laser scanning fluorescence microscope setup of Nikon ECLIPSE Ti (Japan) with an oil dipping objective less (100×). Synthesis of ZIF-8. ZIF-8 was synthesized according to the previously reported literatures with some modification.45 Typically, 150 mg of Zn(NO3)2•6H2O was dissolved in 7 mL methanol, and 350 mg of 2-methylimidazole was also dissolved in another 7 mL methanol. To ensure their thorough dissolution, proper ultrasound was required. Then, the two solutions were mixed under vigorous stirring at room temperature for 5 min. Finally, the obtained nanoparticles were washed three times with methanol and re-dispersed in 14 mL sterile water. The concentration of the obtained ZIF-8 nanoparticles was about 1.0 mg mL-1. Fluorescence Detection of miRNA-21 in vitro. In a typical assay of miRNA-21, 6 µL of DNAzyme strand (1 µM), 5 µL of substrate strand (1 µM), 20 µL of Zn2+ solution (1 mM) and 20 µL of 5×HEPES buffer (125 mM HEPES, 685 mM NaCl, pH 7.4) were added sequentially into 44 µL of sterile water, and the mixture were incubated at 37℃ for 2 h. Subsequently, a series of different concentrations of miRNA-21 (5 µL) was added to the above mixture and then incubated for another 2 h at 37℃. The fluorescence spectra from 555 nm to 750 nm were recorded under the excitation wavelength of 535 nm. For the selectivity assay, single-base, twobase, three-base mismatch stand and other miRNAs were added to the mixture instead of miRNA-21 target to incubate for another 2 h at 37℃. Then, the fluorescence spectra were collected.



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Agarose Gel Electrophoresis. In the gel electrophoresis assay, a mixture containing DNAzyme strand (2.4 µM), substrate strand (2 µM) and Zn2+ solution (1 mM) in 1×HEPES buffer (25 mM HEPES, 137 mM NaCl, pH 7.4) was first incubated at 37℃ for 2 h. Subsequently, miRNA-21 was added to the mixture (2 µM of the final concentration) and then incubated for another 2 h at 37℃. The sample with 1×loading buffer was applied to an agarose gel (4%, w/v). The electrophoresis was carried out in 1×Tris-Borate-EDTA (TBE) buffer (90 mM Tris-HCl, 90 mM boric acid, and 2 mM EDTA, pH 8.0) at 100 V constant voltage for 2 h at room temperature. Cell Culture Conditions. HeLa cells and L02 cells were cultured in RPMI-1640 (GIBCO) with 10% fetal bovine serum (FBS), penicillin (100 units mL-1) and streptomycin (100 µg mL-1) in a humidified atmosphere containing 5% wt/vol CO2 at the temperature of 37℃. MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin (100 units mL-1) and streptomycin (100 µg mL-1). Confocal Fluorescence Imaging of miRNA-21. In a typical fluorescence imaging of miRNA-21, 5 µL of DNAzyme strand (10 µM), 5 µL of substrate strand (10 µM) and 20 µL of 5×HEPES buffer (125 mM HEPES, 685 mM NaCl, pH 7.4) were added sequentially into 60 µL of sterile water, and the mixture were incubated at 37℃ for 2 h. Subsequently, 10 µL of ZIF-8 nanoparticles (1 mg mL-1) was added to the mixture and incubated for 30 min at 37℃ to prepare the probe/ZIF-8 nanocomplex. MCF-7 cells (0.5 mL, 1×106 cells mL-1) were seeded in a 20-mm confocal dish and cultivated in 2 mL DMEM containing 10% fetal bovine serum for 24 h at 37℃. The probe/ZIF-8 nanocomplex (100 µL) was added to 900 µL DMEM and incubated with the cells for 3 h at 37℃. Then, the cells were washed three times with 1×PBS (6.7 mM PB, pH 7.4).


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Finally, 10 µL of Hoechast 33342 (1 mg mL-1) was added to 990 µL of 1×PBS and incubated with the cells at 37℃ for 10 min. Fluorescence images were obtained using a confocal laser scanning fluorescence microscope. The expression levels of miRNA-21 in living cells were regulated by adding MicrONTMhsamiR-21-5p mimic or MicrOFFTMhsa-miR-21-5p inhibitor. Typically, 10 µL of mimic (20 mM) or 12.5 µL of inhibitor (20 mM), 60 µL of 1×buffer and 6 µL of transfection reagent were added sequentially to 924 µL of RPMI-1640 containing 10% fetal bovine serum and incubated with HeLa cells for 24 h at 37℃. The subsequent incubation with the probe/ZIF-8 nanocomplex and Hoechast 33342 stain were the same as the mentioned above. Determination of Cell Viability. The cell viability was evaluated by using MTT Cell Proliferation and Cytotoxicity Assay Kit. HeLa cells (5×103 cells mL-1) were seed in 96-well plates in the culture medium for 24 h under 5% wt/vol CO2 atmosphere. Subsequently, 10 µL of ZIF-8 nanoparticles (1.0 mg mL-1) was added to each well containing 990 µL of culture medium. After incubation for different times, 10 µL of MTT (5 mg mL-1) were added to each well and incubated for 4 h in a humidified incubator at 37℃ with 5% wt/vol CO2. Then, 100 µL of Formazan solution was added to each well for dissolving crystals formed in living cells. After incubation for another 4 h, the absorbance at 490 nm was measured on a microplate reader to calculate the cell viability.

RESULTS AND DISCUSSION



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Design and Detection Principle. In this work, the 8-17 DNAzyme was chosen for constructing the fluorescence sensor because Zn2+ was the effective cofactor for the 8-17 DNAzyme and the 8-17 DNAzyme was well studied previously to identify the conserved sequence within its catalytic core.46,47 A substrate strand containing a complementary segment to the target miRNA-21 was designed, and the complementary segment was also partly complementary to the DNAzyme strand as shown in Scheme 1. The DNAzyme strand was attached with a fluorophore (Cy5) at the 5’ end, and the substrate strand was labeled with a fluorophore (Cy3) at the thymidine near to the site of Cy5. The DNAzyme strand and the substrate strand first formed a stable DNAzyme-substrate duplex probe. The fluorescence resonance energy transfer (FRET) between fluorophore Cy3 and Cy5 occurred due to the close proximity between the two fluorophores in the duplex. Subsequently, the DNAzyme-substrate duplex probe was adsorbed on the surface of ZIF-8 nanoparticles through electrostatic interaction. After cellular uptake, the probe/ZIF-8 nanocomplex was degraded in acidic environment of endosome, releasing duplex probes and Zn2+. The DNAzyme cleaved the substrate into two fragments in the presence of Zn2+. The FRET still occurred because the two fragments were designed to be stable enough by binding with the DNAzyme strand. In the presence of target miRNA-21, the target hybridized with the complementary fragment of the substrate strand, enabling the dissociation of the substrate fragment from DNAzyme-substrate duplex. The dissociation resulted in significant increase in the distance between the two fluorophores. As a result, the FRET was blocked and the fluorescence signal changed. To the best of our knowledge, this was the first attempt to deliver nucleic acid probe to living cells by using ZIF-8 nanoparticles for constructing a ratiometric fluorescence sensor on the basis of DNAzyme, which was


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activated by the metal ions releasing from nanoparticles in living cells. Compared with the conventional double-stranded probe design, this DNAzyme-based design can improve significantly the probe stability and reduce the background signal. In addition, this ratiometric fluorescence imaging strategy can achieve simultaneous imaging of the probe delivery efficiency and the expression levels of miRNA-21 target, avoid the false results due to the failure in probe delivery, and afford a higher imaging contrast. Synthesis and Characterization of ZIF-8. The ZIF-8 nanoparticles were prepared by mixing zinc nitrate and 2-methylimidazole in methanol. The transmission electron microscopy (TEM) image showed that these nanoparticles displayed uniform and monodispersed hexagonal plate-like morphology with mean sizes of approximately 80 nm (Figure 1A). The X-ray diffraction (XRD) analysis indicated that the ZIF-8 nanoparticles possessed identical peaks assigned to a standard ZIF-8 crystal structure (Figure 1B). Zeta potential measurement showed that the surface charge of ZIF-8 nanoparticles was positive, which might be attributed to the abundant Zn2+ on the surface of ZIF-8 (Figure 1C). This result implied that the ZIF-8 could adsorb negatively charged nucleic acid probes through electrostatic interaction. Next, we investigated the adsorption capability of the ZIF-8 nanoparticles to the nucleic acid probe. The DNAzyme strand and the substrate strand were first incubated to form a stable DNAzyme-substrate duplex probe, and then the ZIF-8 nanoparticles were added to adsorb probes followed with washing by centrifugation. Zeta potential of the ZIF-8 nanoparticles changed from positive to negative after incubation with nucleic acid probes, strongly demonstrating the adsorption of the probes on the surface of ZIF-8 (Figure 1C). Dynamic light scattering (DLS) measurements gave the average diameters of 105 ± 12 nm and 164 ± 19 nm for ZIF-8 and



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DNA/ZIF-8, respectively (Figure S1). The increase in the DLS diameter for DNA/ZIF-8 was consistent with the presence of DNA probes on the ZIF-8 surface. The DNA probe loading did not change the ZIF-8 crystal structure as demonstrated by XRD analysis (Figure 1B). The probe loading efficiency was quantitatively examined by determining the fluorescence intensity of the dye-labeled DNA duplex probes before and after incubation with ZIF-8 nanoparticles (Figure S2). The loading efficiency was determined to be as high as 93.41 ± 1.8%, corresponding to the adsorption of about 2.81×1012 DNA probes per 1 µg of ZIF-8 nanoparticles. As a result of steric hindrance effect on surfaces, ZIF-8 nanoparticles could protect DNA probes from DNase I degradation: the fluorescence of the duplex probes formed by FAM-labeled substrate strands and Dabcyl-labeled DNAzyme strands increased rapidly upon incubating with DNase I, while no obvious fluorescence changes could be observed for the DNA probe/ZIF-8 nanocomplex under the same condition (Figure 1D). ZIF-8 possesses unique pH-responsive degradation property. Here, we also interrogated this property by using TEM. As can be seen from Figure 1A, compared with the ZIF-8 nanoparticles under pH 7.4 HEPES buffer for 2 h, the diameter of the ZIF-8 nanoparticles significantly decreased when incubated with pH 5.5 HEPES buffer for 15 min, and the morphology also changed obviously. This result suggested that the ZIF-8 possessed pH-sensitive decomposition property which could be exploited as an intracellular nucleic acid probe delivery vehicle to release probes under mild acidic condition of endosome. In addition, because the designed ratiometric fluorescence imaging strategy was based on DNAzyme that need Zn2+ as an effective cofactor, the release of Zn2+ during the ZIF-8 decomposition was also investigated by using inductively coupled plasma mass spectrometry (ICP-MS). Under pH 5.5 conditions, the releasing


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Zn2+concentration increased remarkably with increasing incubation time, and an almost complete release of Zn2+ could be obtained after 3 h (Figure 1E, Table S2). On the contrary, no obvious changes in Zn2+concentration could be observed at pH 7.4. Under the experimental conditions used in this work, the intracellular Zn2+ concentration after incubation with ZIF-8 nanoparticles was estimated to be 17.18 mM, which was sufficient to make DNAzyme work efficiently in living cells. Detection of miRNA-21 in vitro. Next, we investigated the feasibility of the DNAzyme-based ratiometric fluorescence strategy for the detection of miRNA-21 in vitro. After the DNAzyme strand and substrate strand were mixed and incubated to form stable double-stranded probes, the FRET from Cy3 to Cy5 occurred, as demonstrated by the obvious Cy5 emission peak at 670 nm under Cy3 excitation wavelength at 535 nm (Figure 2A, purplish red curve). In the presence of Zn2+, although the DNAzyme cleaved the substrate into two fragments, the FRET still took place as the two fragments were designed to be stable enough by binding with the DNAzyme strand (Figure 2A, brown curve). When the target miRNA-21 was introduced to the system with Zn2+, the target hybridized with the complementary segment of the substrate strand, resulting in the dissociation of the Cy3-labeled substrate fragment from DNAzyme-substrate duplex probes. The FRET was blocked, and an increased Cy3 emission peak at 565 nm was observed accompanied with a decreased Cy5 emission at 670 nm (Figure 2A, green curve). The ratio of fluorescence intensity at Cy3 to Cy5 (RCy3/Cy5) could be used as the output signal of the ratiometric fluorescence detection. In the absence of Zn2+, the introduction of the target miRNA-21 to the DNAzyme-substrate duplex probes could not lead to the dissociation of the Cy3-labeled substrate strand because the DNAzyme-substrate duplex probes were designed to be extremely



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stable and the FRET still occurred (Figure 2A, blue curve). The sensor (DNAzyme hybridized with substrate) and cleaved sensor were very stable in cell culture medium and no dissociation or degradation took place (Figure S3). In addition, the detection principle was further confirmed by using agarose gel electrophoresis experiment (Figure 2B). The band of DNAzyme-substrate duplex gave no obvious changes after addition of Zn2+, suggesting that no DNA strand dissociated from DNAzyme-substrate duplex although the DNAzyme cleaved the substrate into two fragments (Figure 2B, lane 5). The incubation of target miRNA-21 with DNAzyme-substrate duplex produced a band with higher molecular weight compared with DNAzyme-substrate duplex, which might be attributed to the formation of miRNA-21-DNAzyme-substrate threestranded structure (Figure 2B, lane 6). In the presence of Zn2+, DNAzyme cleaved substrate and produced a nick at rA site, which resulted in the dissociation of the hybrid formed by miRNA-21 and complementary fragment in substrate strand from DNAzyme-substrate duplex. As a result, a band with lower molecular weight compared with DNAzyme-substrate duplex could be observed (Figure 2B, lane 7). These results were consistent with those obtained by fluorescent spectrometry. Moreover, the agarose gel electrophoresis image was also performed using EB, Cy3 and Cy5 (Figure S4), and the results were similar as those obtained in Figure 2B. To obtain the best FRET efficacy, a series of different ratios of substrate strand to DNAzyme strand was used to form DNAzyme-substrate duplex probes. A relatively little ratio of fluorescence intensity at Cy3 to Cy5 (RCy3/Cy5) was obtained when using 1:1.2 ratio of substrate strand to DNAzyme strand (Figure S5). In addition, the reaction time was also investigated. The ratio of fluorescence intensity of Cy3 to Cy5 (RCy3/Cy5) increased remarkably with the reaction time after addition of target miRNA-21, and obtained a maximal value at 2 h (Figure S6). On the


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contrary, no obvious changes in RCy3/Cy5 could be observed when a non-target miRNA-16 was added (Figure S7). Therefore, 1:1.2 ratio of substrate strand to DNAzyme strand and a reaction time of 2 h were selected in the following experiments. Under the optimized experimental conditions, the performance of the ratiometric fluorescence method was interrogated. With the increase in the target miRNA-21 concentration from 0 to 100 nM, the fluorescence intensity of Cy3 emission peak at 565 nm increased gradually, while the fluorescence intensity of Cy5 emission peak at 670 nm decreased (Figure 3A). The ratio of fluorescence intensity of Cy3 to Cy5 (RCy3/Cy5) increased linearly with the miRNA-21 concentration in the range of 2-60 nM (Figure 3B), and the detection limit was estimated to be 0.68 nM (in terms of the rule of 3 times deviation over the blank response), which was comparable to those obtained by existing nanomaterial based methods for intracellular miRNA imaging.48,49 The specificity of the ratiometric fluorescence method was also investigated. As can be seen from Figure 4, single-base mismatched DNA strand seriously affected the detection of target miRNA-21, and two-base mismatched DNA strand also slightly interfered with the target detection. Other than that, no significant influence was observed for three-base mismatched DNA strand and other miRNAs. In addition, there was no obvious signal change after addition of target miRNA-21 to DNAzyme-mismatched substrate duplex probes. These results indicated that the proposed DNAzyme-based ratiometric fluorescence strategy provided good specificity for the target miRNA-21 detection. Imaging of miRNA-21 in Living Cells. Having demonstrated the capability of the proposed method to detect miRNA-21 in vitro, we then explored the feasibility of the DNA probe/ZIF-8



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nanocomplex for imaging of intracellular miRNA-21 expression level. The cytotoxicity of the ZIF-8 nanoparticles was first evaluated by using MTT cell proliferation and cytotoxicity assay. The cell viability still remained 90% after the HeLa cells were incubated with 10 µg mL-1 of ZIF8 nanoparticles for 4 h (Figure S8), suggesting the low cytotoxicity and excellent biocompatibility of the ZIF-8. This exceptional property could be attributed to the low cytotoxicity of Zn2+ and 2-methylimidazole, the products of ZIF-8 decomposition under acidic condition that commonly existed in organism. Next, we investigated the delivery efficiency of the ZIF-8 based nanocarrier platform and intracellular location of DNA probes. HeLa cells were incubated with ZIF-8 nanoparticles adsorbed with Cy5-labeled DNAzyme strands for 3 h and then co-stained with lyso@tracker and Hoechast 33342. As can be seen from the fluorescence images shown in Figure S9, a large number of DNA probes escaped successfully from lysosome and distributed uniformly in the whole cytosol. This exceptionally high cellular uptake might benefit from the abundant positively charged Zn2+ on the ZIF-8 surface, which could induce efficient endocytosis through electrostatic interaction with the negatively charged cell membrane. In addition, the protonated positively charged

2-methylimidazole, produced

by ZIF-8

decomposition

in acidic

endosome/lysosome, also facilitated the escape of DNA probes from endosome/lysosome to cytoplasm. Taken together, ZIF-8 represented an intriguing nanocarrier material for efficient delivery of nucleic acid probes to cytoplasm, promoting the development of various nucleic acidbased assays in living cells. MCF-7 cells, which over-expressed miRNA-21,50 were chosen to estimate the capability of the DNA probe/ZIF-8 nanocomplex to image intracellular miRNA-21 expression level. After


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incubation with 10 µg mL-1 DNA probe/ZIF-8 nanocomplex for 3 h, MCF-7 cells gave bright green fluorescence and negligible red fluorescence (Figure 5A a), indicating that intracellular miRNA-21 hybridized with the complementary segment of substrate strands and led to dissociation from DNAzyme-substrate duplex probes after DNAzyme cleaved substrate to two fragments. To demonstrate that the green fluorescence indeed resulted from the hybridization between miRNA-21 and the substrate strands, a control experiment was designed by using a substrate strand with non-complementary sequence to construct DNAzyme-substrate duplex probes. After incubation with MCF-7 cells, only bright red fluorescence could be observed and no appreciable green fluorescence could be identified (Figure 5A b), suggesting that the dissociation of the DNAzyme-substrate duplex probes did not take place because the miRNA-21 could not hybridize with the non-complementary substrate strands. Moreover, the DNA duplex probes with complementary sequence were also transfected into living cells by using the highly efficient transfection reagent, and only red fluorescence could be obtained (Figure 5A c). This result implied that intracellular Zn2+ in physiological concentrations could not make DNAzyme work efficiently without the supplement of Zn2+ through the decomposition of ZIF-8. As mentioned above, the intracellular Zn2+ concentration after incubation with ZIF-8 was estimated by ICP-MS to be 17.18 mM, sufficient to make DNAzyme work efficiently in living cells. All of these results in living cells were consistent with those obtained in vitro by fluorescent spectrometry, manifesting the excellent specificity and reliability of the proposed method. In addition, compared with “turn-on” fluorescence strategy, the ratiometric fluorescence imaging method possessed two significant advantages: first, the ratiometric method could image the delivery efficiency of the probes as indicated by the bright red fluorescence in Figure 5A b and c,



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avoiding the false negative results caused by the failure in probe delivery; second, the inappreciable green fluorescence signal in control experiments of Figure 5A b and c indirectly confirmed that the DNA probes remained intact in the cytoplasm, circumventing the false positive signal resulted from degradation by cellular nuclease. In addition to MCF-7 cells, the proposed method was also applied to image other types of cell lines with different miRNA-21 expression levels including HeLa and L02 cells (Figure 6A). The bright fluorescence signals in three types of cells revealed high delivery efficiency of ZIF-8 nanocarrier in various types of cells. MCF-7 cells exhibited the brightest green fluorescence and L02 cells displayed the brightest red fluorescence, indicating that MCF-7 cells gave the highest expression level of miRNA-21 whereas L02 cells had the lowest expression. These results were consistent with those previously reported for miRNA-21 expression levels in these cells.18,51 Moreover, the ratios of green-to-red fluorescence were also correlated with the relative expression levels of miRNA-21 (Figure 6B), which gave the images with higher contrast and made us easily distinguish cancer cells from normal cells. As a result, the proposed ratiometric imaging method provided a powerful tool for cancer diagnosis. The fluctuation of miRNA-21 expression levels could also be imaged by the proposed method. MicrONTMhsa-miR-21-5p mimic, small double-stranded RNAs mimicking miRNA-21 and enabling up-regulation of miRNA-21 activity,52 and MicrOFFTMhsa-miR-21-5p inhibitor, small single-stranded RNAs designed to specifically bind to and selectively decrease active concentration of miR-21,52 were used to treat HeLa cells to regulate the miRNA-21 expression levels. As can be seen from Figure S10, miRNA-21 mimic treated HeLa cells showed a brighter green fluorescence whereas miRNA-21 inhibitor treated HeLa cells gave a brighter red


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fluorescence compared with untreated HeLa cells, which were consistent with the fact that miRNA-21 mimic could up-regulate miRNA-21 expression whereas miRNA-21 inhibitor could down-regulate miRNA-21 expression. Likewise, the ratiometric images exhibited obvious color changes depending on the relative expression levels of miRNA-21. These results implied that the proposed method hold great potential in the miRNA-associated biological study.

CONCLUSIONS This work adopted for the first time ZIF-8 as a nanocarrier to efficiently deliver nucleic acid probe to living cells and developed a novel ratiometric fluorescence strategy based on DNAzyme for miRNA-21 imaging. The abundant Zn2+ on the ZIF-8 surface could bind a large number of nucleic acid probes through electrostatic interaction and promote efficient endocytosis of nanoparticles. After cellular uptake, the unique pH-sensitive degradation property of ZIF-8 resulted in the release of nucleic acid probes, Zn2+ and 2-methylimidazole in the acidic endosome/lysosome, and the releasing protonated 2-methylimidazole facilitated the escape of nucleic acid probes from endosome/lysosome to cytoplasm. Therefore, ZIF-8 nanocarrier represented an efficient delivery platform. Moreover, the releasing Zn2+, as an effective cofactor for 8-17 DNAzyme, was exploited to develop a ratiometric DNAzyme-based imaging method, providing a new design strategy for nucleic acid assay in living cells. The proposed method has been applied to image miRNA-21 expression levels in MCF-7, HeLa and L02 cells with high contrast and reliability. The fluctuation of miRNA-21 expression level induced by miRNA-21 mimic or inhibitor could also be monitored through the obvious imaging color change. As a



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result, the proposed method holds great potential in cancer diagnosis and miRNA-associated biological study.

ASSOCIATED CONTENT Supporting Information Additional figures and tables as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. DNA sequences, ICP-MS, DLS results, loading efficiency, fluorescence spectras, gel images, cell viability, colocalization confocal imaging and adjusting expression levels of miRNA-21

AUTHOR INFORMATION Corresponding Author * Phone/Fax: +86-731-88821916. E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21525522) and the Foundation for Innovative Research Groups of NSFC (Grant 21521063).


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Scheme 1. Illustration of the design and sensing principle of the ratiometric fluorescence imaging strategy for microRNA-21 based on ZIF-8.


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Figure 1. (A) TEM images of ZIF-8 nanoparticles at pH 7.4 for 2 h (a) and pH 5.5 for 15 min (b). (B) PXRD patterns of ZIF-8 (red), DNA/ZIF-8 (blue), and simulated ZIF-8 (black). (C) Zeta potential of ZIF-8 (red) and DNA/ZIF-8 (black). (D) Fluorescence changes with time of the free duplex DNA probes (black) and DNA probe/ZIF-8 nanocomplex (red) after addition of DNase I. (E) Zn2+ releasing percent with time at pH 5.5 (black) and pH 7.4 (red). A

B

D

a

b

C

E



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Figure 2. (A) Fluorescence spectra of 50 nM Cy5-labeled DNAzyme strand (black), 50 nM Cy3-labeled substrate strand (red), double-stranded probes formed by 50 nM substrate and 50 nM DNAzyme (purplish red), 50 nM double-stranded probes with 0.2 mM Zn2+ (brown), 50 nM double-stranded probes and 50 nM miRNA-21 target (blue), and 50 nM double-stranded probes, 0.2 mM Zn2+ and 50 nM miRNA-21 target (green). (B) The agarose gel electrophoresis image.

A

B


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Figure 3. (A) Fluorescence spectra of the DNAzyme-substrate double-stranded probes in the presence of Zn2+ after addition of a series of different target miRNA-21 concentrations. The excitation wavelength was set at 535 nm. (B) The plot of the ratio of fluorescence intensity of Cy3 to Cy5 (RCy3/Cy5) versus the target miRNA-21 concentration. Insert: Linear correlation of the ratio (RCy3/Cy5) against concentrations of miRNA-21 target. Error bars represented standard deviation of three repetitive assays. A

B

Figure 4. (A) Fluorescence spectra of the DNAzyme-substrate double-stranded probes in the presence of Zn2+ (purplish red) after addition of target miRNA-21 (yellow), single-base mismatched strand (light blue), two-base mismatched strand (brown), three-base mismatched strand (blue), non-target miRNA-16 (red), non-target miRNA-26a (green), and DNAzymemismatched substrate double-stranded probes in the presence of Zn2+ after addition of target miRNA-21 (black). (B) The ratio of fluorescence intensity of Cy3 to Cy5 (RCy3/Cy5) after addition of various analytes. A


B


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Figure 5. (A) Fluorescence imaging of MCF-7 cells. (a) Incubated with 10 µg mL-1 DNA probe/ZIF-8 nanocomplex for 3 h; (b) incubated with 10 µg mL-1 ZIF-8 nanocomplex adsorbed with non-complementary substrate strand-formed DNA probes for 3 h; and (c) incubated with DNA probe/transfection reagent complex for 2 h. The excitation wavelength was set at 560 nm, and fluorescence signals were collected at 570-620 nm for green channel and 663-738 nm for red channel. Scale bar: 20 µm. (B) The ratio of fluorescence intensity of Cy3 to Cy5 (RCy3/Cy5) at different treatments. A B


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Figure 6. (A) Fluorescence imaging of MCF-7 cells (a), HeLa cells (b) and L02 cells (c) incubated with 10 µg mL-1 DNA probe/ZIF-8 nanocomplex for 3 h and Hoechast 33342 for 10 min. The excitation wavelength was set at 560 nm, and fluorescence signals were collected at 570-620 nm for green channel and 663-738 nm for red channel. Scale bar: 20 µm. (B) The ratio of fluorescence intensity of Cy3 to Cy5 (RCy3/Cy5) of different cells. A B



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For TOC Only


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Nanoscale Zeolitic Imidazolate Framework-8 for Ratiometric

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